1. Field of the Invention
The present invention relates to methods for the cryopreservation of plant cells and to methods for the recovery of plants cells which have been cryopreserved. The invention also relates to plants, viable plant cells and plant cells cultures which have been successfully recovered from cryopreservation.
2. Description of the Background
Cryopreservation is based on the reduction and subsequent arrest of metabolic functions of biological material stored at ultra-low temperatures. Cryogenic preservation of plants and plant cells for extended periods without genetic change and the subsequent recovery of normal plant cells with unaltered characteristics and biosynthetic ability has important implications in plant breeding, biomedical research and genetic engineering. At the temperature of liquid nitrogen (-196.degree. C.) almost all metabolic activities the cell ceases and cells can be maintained in this suspended, but viable state for extended periods.
Plant cells are cryopreserved to avoid loss by contamination, to minimize genetic change in continuous cell lines, and to avoid aging and transformation in finite cell lines. Traditional methods for preservation of a desirable plant characteristic involve establishment of colonies of plants in the field because many plants do not breed true from seeds. These field plant depositories demand large inputs of labor and land and incur high risks of loss due to weather, disease or other hazards. An alternative to a field colony is the establishment of an in vitro collection of plant tissue under normal or limited growth conditions. For long-term storage, elimination of routine subculturing is desirable because of concerns with mutation, contamination, labor cost and risk of human error associated with tissue culture.
Most biological materials, including plants, cannot survive freezing and thawing from cryogenic temperatures without cryoprotective agents and procedures. A number of cryopreservatives possess properties which can protect a cell from the damaging effects of cryogenic freezing. The essence of cryopreservation is to effect cell dehydration and concentration of the cytosol in a controlled and minimally injurious manner so that ice crystallization in the cytosol is precluded or minimized during, for example, quenching in liquid nitrogen.
In conventional cryopreservation procedures, cell dehydration is effected by freeze-induced concentration of the suspending medium. Deleterious effects of dehydration are mitigated by the presence of cryoprotective agents. Specimens such as cells and organs are equilibrated in a solution containing a cryopreservation agent such as dimethylsulfoxide (DMSO) or ethylene glycol. The suspension is cooled and seeded with an ice crystal at a temperature slightly below its freezing point. The suspension is cooled again at an optimum rate to an intermediate sub-zero temperature such as between about -30.degree. C. and about -40.degree. C. and finally quenched in liquid nitrogen.
While routine cryogenic preservation of microorganisms, zygotes and animals derived from zygotes is possible, the cryopreservation of plant cells is far from routine and often, different protocols for individual species of plants are necessary.
Taxus trees produces taxol, a diterpenoid alkaloid originally isolated from the bark of the Pacific yew, Taxus brevifolia (M. C. Wani et al., J. Am. Chem. Soc. 93:2325-27, 1971). Experiments have demonstrated that this compound effectively inhibits the polymerization of microtubules of mammalian cells without undue toxicity and, as such, is an effective anti-tumorigenic agent. Clinical trails identified taxol as extremely effective against refractory ovarian, breast and other cancers. As such, taxol is a breakthrough in chemotherapy because of its rather unique, but basic mechanism of action which is fundamentally distinct from that of conventional chemotherapeutic agents (L. A. Rowinsky et al., J. Natl. Cancer Instit. 82:1247-59, 1990).
The most daunting variable in the taxol equation so far is supply. It takes three to six, 100 year old Pacific yews to treat one patient because average yields of taxol are low (Witherup et al., 1990). The production of an amount of taxol needed for treatment and testing will require the destruction of tens of thousands of yews. The yew population has been rendered nearly extinct by logging and as the number of Pacific yews dwindles, medical research must look for other forms of supply for taxol. The usefulness of taxol, as well as many other compounds which may be propagated or harvested in plant cells, has fueled an interest in culturing taxus and other plant cells.
The culturing of plant cells for their biosynthetic ability poses special problems for current technology. Prolonged culturing of plant cells often results in a loss of biosynthetic ability which had been present in the original isolates (Dhoot et al., Ann. Bot. 41:943-49, 1977; Barz et al., Ber. Dtsch. Bot. Ges. 94:1-26, 1981). Phenotypic alterations also arise which further complicate cell culturing. A protocol for freezing plant cells, especially taxus cells, is an important step in the development of biosynthetic methods for production of useful plant alkaloids such as taxol.